Perspectives on Past Practices and Future Trends in Toxicologic Neuropathology

Abstract

Dr. Peter S. Spencer, a pioneering neurotoxicologist of international renown, delivered the keynote address at the 2010 Joint Scientific Symposium of the Society of Toxicologic Pathology (STP) and the International Federation of Societies of Toxicologic Pathologists (IFSTP). He has made many landmark discoveries during his four-decade career. Dr. Spencer’s address communicated several fundamental principles of past and present toxicologic neuropathology research, and he also predicted future trends in the field. First, classic approaches to toxicologic neuropathology emphasized morphologic techniques such as light microscopic and ultrastructural assessment. However, neuropathology methods alone rarely reveal the mechanism(s) and etiology of neurotoxic conditions, so neurotoxicity problems are now being investigated using a multidisciplinary approach in which neuropathologic assessment is but one component of the analysis. The two primary trends for future toxicologic neuropathology investigations, in both animals and humans, will be an increased use of noninvasive neural imaging and greater preference for in situ molecular (“omic”) methods, which provide functional information in a structural context. These trends will significantly enhance the ability of scientists to translate animal data to human situations, thereby improving our understanding of disease mechanisms and facilitating efforts to design new therapies for neural diseases.

Peter Spencer, PhD, FRCPath, delivered the keynote address at the 2010 symposium on “Toxicologic Neuropathology,” which was sponsored jointly by the Society of Toxicologic Pathology (STP) and the International Federation of Societies of Toxicologic Pathologists (IFSTP). Dr. Spencer’s extensive career as a pioneer neurotoxicologist, in which he has employed investigative techniques ranging from single nerve fiber electron microscopy to clinical research in the jungles of New Guinea, exemplifies the importance of this scientific discipline to individual and collective human health. Moreover, four decades of continuous effort in this field have given him a matchless perspective on past practices in toxicologic neuropathology, as well as a firm understanding of the interdisciplinary endeavors that will sustain and expand the roles of toxicologic neuropathologists in the future. Dr. Spencer has documented his toxicologic neuropathology efforts in over 400 scholarly contributions, half representing original research in peer-reviewed journals and the balance consisting of review articles, commentaries, book chapters, and books. Signal contributions to the field include his classic texts Experimental and Clinical Neurotoxicology (Spencer and Schaumburg 1980; Spencer et al. 2000 [with the introductory chapters of the latter edition available in a Chinese translation]) and Disorders of Peripheral Nerves (Schaumburg et al. 1983). His body of work has significantly advanced our understanding of neuropathology and neurotoxicology in the peripheral (PNS) and central (CNS) nervous systems, especially with respect to degenerative diseases, as well as the structural and functional processes that occur in health and in regenerative and aging states.

Past and Current Practices in Toxicologic Neuropathology

At the start of Dr. Spencer’s research career, toxicologic neuropathology consisted chiefly of conventional morphologic studies emphasizing light microscopic assessment of formalin-fixed tissues. Typical specimens were brains, ganglia, and peripheral nerve biopsies from human patients, as well as comparable tissues from animal models with spontaneous or induced (using chemicals or surgical manipulations) neural diseases that exhibited clinical signs similar to some human condition. The biochemical basis of neural diseases usually was explored by a combination of specialized morphologic methods (stains for particular cell types or neurochemicals) and experimental designs in which a putative neurotoxicant or neuroprotective agent was administered to evaluate lesion progression. Related neurobiological disciplines, such as clinical neurology and electrophysiology, were used to construct a more complete picture of a neurotoxic syndrome. Molecular investigations, including such innovative in situ morphologic methods as immunohistochemistry and in situ hybridization, had not yet been discovered.

Dr. Spencer made numerous pioneering contributions that significantly advanced our understanding of basic neurobiology and toxicologic neuropathology during the first decade of his career. His first publications demonstrated the extraordinary increase in definition and resolution of morphologic detail relative to light microscopy when neural tissues were prepared for electron microscopy using buffered glutaraldehyde and osmium tetroxide fixation (Spencer and Lieberman 1971; Spencer, Peterson et al. 1973; Spencer, Raine et al. 1973). He was one of the first to report a method to tease apart long lengths of individual peripheral nerve fibers while preserving their ultrastuctural features, and the first to investigate their three-dimensional morphology and pathology using light microscopy in combination with both transmission and scanning electron microscopy (Spencer and Lieberman 1971; Spencer and Thomas 1970). His early experiments in which transected myelinated nerves were anastomosed to the cut ends of largely unmyelinated nerves provided the initial evidence for neuronal control of PNS myelination through axon–Schwann cell contact (Weinberg and Spencer 1975). He introduced the surgical creation of a perineurial window, which permitted serial temporal evaluation of demyelination and remyelination (Spencer et al. 1975). He was the first to use organotypic cultures of structurally and functionally integrated neural domains (peripheral nerves, ganglia, spinal cord) to visualize the spatial-temporal evolution of axonal degeneration induced by treatment with various neurotoxicants (Spencer, Peterson et al. 1973; Veronesi et al. 1983). These early studies culminated in a series of landmark papers on the toxicant-induced dying-back process (retrograde axonal degeneration), which demonstrated how the Schwann cell and oligodendrocyte activated a mechanism to sequester and eliminate degenerate organelles from the axoplasm (Politis et al. 1980; Schaumburg et al. 1974; Spencer and Schaumburg 1977a; Spencer and Schaumburg 1977b; Spencer and Thomas 1974; Veronesi et al. 1983)

During this same period, Spencer’s research was a major catalyst for the transition to a more interdisciplinary approach to neurotoxicity assessment. This evolution was required because neuropathology methods alone could not provide firm, direct evidence that a potential neurotoxicant is responsible for inducing a given neurological disease or confirm the mechanisms by which the agent could act at the cell and molecular level. Dr. Spencer’s comparison of animal and human responses to neurotoxic chemicals that induced peripheral neuropathy (e.g., acrylamide, n-hexane, methyl-n-butyl ketone, organophosphates) led him to introduce the concept of “central-peripheral distal axonopathy,” in which long and large-diameter myelinated fibers in peripheral nerves, spinal cord, and other long CNS pathways undergo retrograde axonal degeneration (Spencer and Schaumburg 1977b). His subsequent molecular investigations, comparing the capacities of structurally diverse agents to incite axonal neurofilament accumulation, revealed that systemic intoxication with certain aliphatic (2,5-hexanedione) and aromatic (1,2-diacetylbenzene) γ-diketone hydrocarbons results in CNS and PNS axonopathy, whereas exposure to compounds lacking the γ-diketone arrangement (e.g., 2,4-hexanedione, 1,3-diacetylbenzene) does not (Spencer et al. 1978; Spencer et al. 2002). Spencer’s research also demonstrated that repeated exposure to certain chemicals (acetylethyltetramethyltetralin, musk ambrette) accorded GRAS (“generally recognized as safe”) status caused CNS and/or PNS damage (Spencer et al. 1984; Sterman and Spencer 1981), thereby resulting in their worldwide withdrawal as human food additives. He also confirmed that 3-nitropropionic acid, a mycotoxin, was responsible for an encephalopathy that developed in Chinese children who had consumed fungus-contaminated sugarcane (Ludolph et al. 1991). In sum, Dr. Spencer’s first decade of research led to the discovery and definition of numerous neurotoxic chemicals to which humans were exposed, prompting their removal or regulation, and fostered the standard practice of using active, and inactive, substances from a given compound class as chemically defined tools to probe neural functions, structural lesions, and cellular and molecular mechanisms in laboratory animal species and in vitro models resembling human neurodegenerative diseases.

The subsequent three decades of Spencer's career were engaged in defining new toxicologic neuropathology approaches to better understand and prevent human neurodegenerative diseases. His research focus during this period was shaped by two factors. The first was the realization that neuropathology investigations using in vivo and in vitro animal models of neurotoxic disease represented an indirect, and usually incomplete, means of investigating the etiology of comparable states in human patients, whereas direct examination of the environment and circumstances under which the disease evolved provided a valuable supplement for drawing conclusions. The second factor was Spencer’s frustration at his inability to identify a neurotoxic substance for large brain and spinal cord motor neurons of patients suffering from neurodegenerative conditions. This aggravation led him to investigate neurodegenerative conditions in low-income countries for which dietary neurotoxicants were suspected: lathyrism, a little-known upper motor neuron disease caused by dietary dependence on Lathyrus sativus (grass pea), and amyotrophic lateral sclerosis and parkinsonism dementia complex (ALS/PDC), a prototypical tauopathy that was once widespread in three Pacific island groups where the natives regularly consume cycad seeds but whose incidence has recently declined for unknown reasons. Spencer’s new paradigm for toxicologic neuropathology was to exhaustively evaluate the human conditions using on-site clinical, neurophysiological, and epidemiological methods, as well as laboratory-based morphologic and molecular studies in human neural tissues, before moving on to in vivo and in vitro animal models; in essence, this approach applies Koch’s postulates of bacterial pathogenesis to putative neurotoxic agents. A long series of his papers supports the hypothesis that neuron loss in lathryism is caused by excitotoxicity (reviewed in Spencer 1995), resulting in a self-limiting spastic disease, whereas cycad harbors potent neurotoxins with excitotoxic (β-methylamino-L-alanine [BMAA]) and DNA-damaging (cycasin) properties that appeared to trigger progressive neurodegeneration by an unknown mechanism (reviewed in Spencer and Kisby 1992). In his keynote address, Dr. Spencer noted that a long-term collaborative project designed to dissect intracellular pathways of toxicant-induced neurodegeneration had discovered different patterns of altered gene expression following cycasin treatment in wild-type mice relative to mice lacking a key DNA repair enzyme. Ultimately, this and similar “omics” projects should help to identify molecular mechanisms, and the chemical trigger(s) of prototypical neurodegenerative diseases, with relevance to understanding the etiology and pathogenesis of related tauopathies, including Alzheimer’s disease.

Future Trends in Toxicologic Neuropathology

Taken together, Spencer’s recent approach for investigating human neurodegenerative diseases identifies two major trends for future toxicologic neuropathology investigations: emphasis on acquisition of human data (because of its direct relevance to the understanding and treatment/prevention of neurologic diseases) and reliance on interdisciplinary (and often multi-investigator) assessments. These two trends support two clear predictions regarding innovations that will be adopted by toxicologic neuropathologists in the next ten to twenty years.

The first is that novel morphologic technologies, such as noninvasive imaging, will see increased use as a supplement for—and in some instances as a replacement for—traditional, slide-based neuropathology techniques. Advantages of noninvasive imaging include the ready ability to translate structural and in situ functional findings from animal models (Gabrielson et al. 2011) to humans (Arora et al. 2008; Pogge and Slikker 2004), and the simplicity of performing longitudinal neuropathology studies; a particular benefit will be the ability to follow the progression of neural lesions over time at both the gross and microscopic levels in a single individual. Current efforts are seeking to adapt the quantitative morphometric procedures, used routinely in conventional slide-based toxicologic neuropathology analyses, for use with brain images (Jacobson et al. 2009).

The second prediction is that toxicologic neuropathology data sets will increasingly come to rely on methods that combine conventional morphologic assessment with the simultaneous acquisition of biochemical and/or molecular data. This trend is obvious in the numerous experimental neuropathology reports in which the expression of one or more cell type–specific molecules is evaluated in vivo in animals, or in animal or human neural tissues, following exposure to a neurotoxicant. Furthermore, regulatory agencies currently advocate that product registration packages for chemicals and certain classes of drugs incorporate information regarding a compound’s neurotoxic potential derived using special stains and/or immunohistochemical procedures to assess neuronal and glial populations (Bolon et al. 2006; Bolon et al. 2008). An alternative approach to in situ molecular studies will be to link samples for neuropathology and toxicogenomic analyses so that the impact of multiple genes on disease initiation and progression may be assessed concurrently (Pandiri et al. 2011). Combined structural and functional data of this kind will be essential for extrapolating findings from animals to humans, for understanding disease mechanisms, and for rationally designing treatments that can ameliorate or even prevent neurodegenerative diseases or promote neuroregeneration.

Dr. Spencer is preparing to pass the toxicologic neuropathology baton to a new generation of researchers. The rigorous foundation he laid in the past for using both standard morphological techniques and innovative interdisciplinary approaches will continue to serve as the essential underpinning for toxicologic neuropathology studies in the future.